By using a nucleic acid probe such as an RNA probe that has been subjected to some form of labeling to detect and visualize the pattern of expression of DNA or RNA at a cellular level, a multitude of problems related to vital phenomena can be explained. This type of technique that enables the visualization of gene expression patterns at a cellular level is termed in situ hybridization (ISH), and the labeling methods employed for the probes used in this technique can be broadly classified into “radioactive isotope labeling methods”, “fluorescent antibody labeling methods” and “enzyme antibody labeling methods.” Historically, nucleic acid probes having an introduced radioactive isotope were developed first, but in recent years, restrictions have been introduced relating to the handling of such probes, and therefore fluorescent antibody labeling methods and enzyme antibody labeling methods, which do not require the use of a radioactive isotope element, are attracting much attention.
In these techniques, labeling is performed with an antigen or biotin during preparation of the nucleic acid probe, and following hybridization of the nucleic acid probe with the target nucleic acid, detection is performed by an immunostaining method using an antibody or avidin labeled with an enzyme or a fluorescent material. Enzyme antibody labeling methods, in which a signal amplification effect is achieved as a result of an enzyme reaction, offer superior sensitivity and are therefore currently the most widely used.
Examples of known nucleic acid probes that utilize enzyme antibody labeling methods include antigen-multilabeled nucleic acid probes in which a plurality of nucleotide derivatives that have been modified at the antibody recognition site such as digoxigenin (DIG) are introduced in a random arrangement. Following in situ hybridization of this antigen-multilabeled nucleic acid probe and the target nucleic acid, an antigen-antibody reaction is conducted with an enzyme-labeled antibody that recognizes the antibody recognition site, and detection is performed using a color development reaction that utilizes a hybrid with enzyme alkaline phosphatase. However, there are several problems associated with this method, including the fact that the enzyme-labeled antibody is extremely expensive, the complexity of the operations required for the antigen-antibody reaction, and an increased background reading caused by nonspecific adsorption and the like.
On the other hand, a method is known in which a transglutaminase (TGase) is used to achieve site-specific binding of an exogenous molecule, which is anionic and has a glutamine (Gln) residue that is recognizable by TGase, to a peptide or protein having a lysine (Lys) residue or a primary amine which are recognizable by TGase (for example, see Patent Document 1).